p450S have been thought of as strictly detoxification/hormone synthesizing enzymes in mammals; however, with the identification of more than P450s in mammals, plants, fungi, and bacteria, it has been realized that the role of P450s is much more diverse than had been previously suspected. With the elucidation of the structure of P450cam and of two additional P450s in our laboratory, P450BM-P and P450terp, we have been able to determine which structural elements are highly conserved and which elements are less conserved. These P450's have an extremely high variability in sequence (less than 10% identity); however, there is an overall three dimensional structure, i.e. a structural fold which is similar in P450cam, P450terp, and P450BM-P. The hypothesis around which this project has coalesced is that there is a P450 structural fold, and hence, a specific folding pathway which characterized this superfamily of proteins. The understanding of this structural fold is critical in the efforts to modify P450 enzyme activities to be used in bioremediation and drug synthesis. Therefore, our aims are to establish conditions for folding, unfolding, an heme reconstitution in order to be able to modify substrate specificity and redox partner binding. Specifically, to test our hypothesis we have set forth the following aims; A. Evaluating the folding/unfolding pathway in P450BM-P i. establishment of conditions for denaturation and subsequent renaturation ii. preparation of P$%)BM-P with heme and heme derivatives to probe the substrate/oxygen binding pocket and heme environment. B. Identifying those structural elements which allow for correct folding, for substrate binding, and for redox-partner binding i. synthesis of P450BM-P using synthetic oligonucleotides ii. systematic elimination of structural elements to construct a "minimal" P450 iii. substitution of structural elements from other p450 s to construct hybrid proteins with hybrid activities C. Determining the three dimensional structures of those p450 forms which are of greatest interest/utility i. evaluate the structure of P450BM-P bound to the substrates arachidonic or eicosapenteneoic acid ii. crystallize and determine the structure of those hybrid/mutant P450s which might shed light on the minimal structure of a P450 folding pathway. Circular dichroism spectroscopy will be used to assess the changes in tertiary structure during folding and unfolding of these proteins, while magnetic circular dichroism spectroscopy will be used to examine changes in the ligand state of the heme.